The estimation of the
total amount of phospholipids in a tissue extract must preferably be estimated before any
chromatographic separation. This prevents the inevitable loss of phospholipid molecules on
adsorbents and thus allows accurate estimation of the ratio of cholesterol to
phospholipids in cellular extracts.
We propose below a simple method to estimate rapidly phospholipids in lipid extract and
two methods based on the estimation of the amount of phosphorus in extracts or
chromatographic fractions. A non destructive method base on NMR spectroscopy was
developed for the determination of phospholipids (Hatzakis E et al., J Agric
Food Chem 2008, 56, 6232). This technique, involving a specialized
instrumentation, enables also the determination of the fatty acid pattern of
these phospholipids.

1. Direct estimation of
phospholipids

This colorimetric method is based on the formation of a
complex between phospholipids and ammonium ferrothiocyanate (Stewart JCM, Anal Biochem
1980, 104, 10).

Evaporate completely aliquots of phospholipid solutions in
glass tubes.
Dissolve the phospholipid residue in 2 ml chloroform and add 1 ml of thiocyanate reagent.
Vortex 1 min and centrifuge at low speed, remove the red lower layer (chloroform) with a
Pasteur pipette.
Read the absorbance of this solution at 488 nm and compare with known amounts of a
standard phospholipid solution (range: 10-100 µg per tube).
All phospholipids do not give the same response but this method is useful for measuring
rapidly phospholipids in mixtures before further analyses.

2.
Phospholipid determination by phosphorus assay

Phospholipids in lipid extracts or in chromatographic
fractions are satisfactorily estimated by phosphorus determination through an acidic
digestion. The released inorganic phosphate is reacted with ammonium molybdate, the
complex giving a strong blue color.
Two methods are proposed below according to the sensibility required.

Lipid samples are transferred into clean glass tubes and the
solvent is completely evaporated. Add 0.65 ml perchloric acid and place the tubes
in the heated block for about 30 min or until the yellow color has disappeared. Silica gel
spots may be digested in the same way.
When cool, add to the tubes 3.3 ml water, 0.5 ml of molybdate solution and then 0.5 ml of
ascorbic acid solution. Agitate on a vortex after each addition.
The tubes are placed in a boiling water bath for 5 min.
The absorbance of cool samples (including the standards) are read at 800 nm
Standards (1 to 5 µg P/tube) are diluted in 3.3 ml water and 0.65 ml perchloric acid.
Digestion is not necessary before adding reagents.
Classically, 5 µg P give an absorbance of 0.9
The amount of phospholipids is calculated directly on a molar basis from the amount of P
and on a weight basis after multiplying the amount of P by 25.

In the presence of silica gel (scraped from TLC plates), centrifuge the tubes at low speed
and remove the colored solution before measuring its absorbance.
As the knowledge of the phospholipid content in a vegetable oil is a necessity
to grade the quality of the oil and examine the efficacy of the refining process,
several methods have been proposed for the oil phosphorus content. An
improvement of the AOCS official method Ca 12-55 for the oil phosphorus content
has been described (Chen B et al., Eur J Lipid Sci Technol 2014, 116, 548).

B. Phosphorus assay for amounts lower than 1 µg (25 µg
phospholipids)

An improved method was developed for the determination of
lipid phosphorus in the sub µg range (Zhou et al., J Lipid Res 1992, 33, 1233).
This method is based on the formation of a complex between phosphomolybdenum and malachite
green and its resultant shift in absorption maximum. It is convenient for the
determination of phospholipid distribution after TLC.

Apparatus:

Electrically heating block set at
180°C.
Glass tubes (to be centrifuged) washed before use in water acidified with conc. nitric
acid (about 1% final) and dried in an oven at 100°C.

Lipid samples are transferred into clean glass tubes and the
solvent is completely evaporated. Add 0.2 ml perchloric acid and place the tubes in
the heated block for 30-60 min or until the yellow color has disappeared.
When cool, add to the tubes 0.2 ml water and 2 ml of working solution. Agitate on a vortex
and measure the absorbance after 20 min at 660 nm..
Standards (0 to 0.2ml) are diluted to 0.2 ml before adding 0.2 ml perchloric acid.
Classically, 0.6 µg P give an absorbance of 1.

B. Lipid samples with silica gel (scraped spots)

Scraped spots are transferred into clean glass tubes, add .2
ml water and 0.4 ml perchloric acid. Heat at 180°C. When cool, centrifuge at low speed
and collect 0.2 ml of the supernatant for phosphorus determination as in A. Multiply the
measured value by 2 to get the total amount present in the tube.

Comments :

A modified version of the malachite procedure was reported to be
relevant for the determination of total phosphorus in vegetal oils (Szydlowska-Czerniak
A et al., Food Chem 2003, 81, 613). After mineralization of oil samples (up
to 26 g) with MgO at 800°C, the malachite green method allowed the estimation
of the very low levels of lipidic phosphorus found in refined oils (about 2 mg
per Kg).

3 - Estimation of phospholipids from fatty acid
amounts

A quantitative determination of the amount of
fatty acids in a phospholipid sample enables a more precise estimation of the
amount of phospholipid than with the phosphorus amount.
To know that amount, the fatty acid weight must be multiplied by 1.3.
When a definite phospholipid class is analyzed, the value of the coefficient
must be adapted to that class. The table below gives these values for the
main phospholipid classes :

Phosphatidylcholine

1.36

Phosphatidylethanolamine

1.26

Phosphatidylserine

1.33

Phosphatidylinositol

1.45

Sphingomyelin

2.63

Cardiolipin

1.43

DETERMINATION OF THE AMOUNT

OF PLASMALOGENS

Three approaches are classically used to quantify plasmalogens
(aldehydogenic lipids), namely the estimation of long-chains aldehydes released through an
acidic treatment, the determination of the vinyl ether content, and the HPLC
separation of intact plasmalogens.

1. Long-chain aldehydes in free or bound forms may be estimated colorimetrically by conversion to nitrophenylhydrazones
or by GLC of the dimethyl acetal derivatives.

2. The vinyl ether content of a phospholipid sample can be determined by the iodine uptake
method as it was described in its last modification by Gottfried et al (J Biol Chem
1962, 237, 329). The specificity of the determination is based on the more rapid
interaction of iodine with vinyl ethers than with olefinic double bonds.

Evaporate an aliquot of lipid solution (50-100 nmol) in a
glass tube and dissolve in 0.5 ml methanol. Add 0.5 ml of working solution and mix for 30
min at room temperature.
Add 4 ml of 95% ethanol and read the absorbance at 355 nm against a blank in which 0.5 ml
of 3% KI is added to the sample instead of the iodine solution. Run also a control tube in
which the lipid sample is omitted. The molar extinction coefficient is about 27 500.

A highly sensitive determination of plasmalogens based on HPLC separation of
molecules labeled with radioactive iodine (125I) was described (Maeba
R et al., Anal Biochem 2004, 331, 169). The method sensitivity is more
than 1000-fold higher than the classical determination with nonradioactive
iodine.